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The mammalian leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family consists of three different members, LRIG1, LRIG2, and LRIG3. These genes are expressed in all human and mouse tissues analyzed to date. All LRIG proteins share similar and evolutionary conserved structural domains including a leucine-rich repeat domain, three immunoglobulin-like domains, a transmembrane domain, and a cytosolic tail. Since the discovery of this family, around 20 years ago, various research groups have shown the importance of this family in cancer biology and prognosis. The aim of this thesis was to further investigate the role of LRIG1 and LRIG2 in cancer.

To investigate the roles of LRIG1 and LRIG2 in physiology and gliomagenesis, we generated Lrig1- and Lrig2-deficient mice and induced platelet-derived growth factor B (PDGFB)-driven gliomagenesis. We studied the effects of Lrig2 ablation on mouse development and survival and investigated if the ablation of Lrig1 or Lrig2 affects the incidence or malignancy of induced gliomas. We also investigated if Lrig2 ablation affects Pdgfr signaling in mouse embryonic fibroblasts (MEFs). Additionally, we analyzed the effects of ectopic LRIG1 expression in human primary glioblastoma cell lines TB101 and TB107, in vivo and in vitro. We reported no macroscopic anatomical defect but reduced growth and increased spontaneous mortality rate in Lrig2-deficient mice. However, the Lrig2-deficient mice were protected against the induced gliomagenesis. Lrig2-deficient MEFs showed faster kinetics of induction of immediate-early genes in response to PDGFB stimulation, whereas the phosphorylations of Pdgfra, Pdgfrb, Erk1/2, and Akt1 appeared unaltered. Lrig1-heterozygote mice showed a higher incidence of high-grade tumors (grade IV) compared to wildtype mice, demonstrating a haploinsufficient function of Lrig1. LRIG1 overexpression suppressed TB107 cell invasion in vivo and in vitro, which was partially mediated through the suppression of the MET receptor tyrosine kinase.

To identify LRIG1-interacting proteins, we used the yeast-two hybrid system and data-mined the Bio-Plex network of high throughput protein-protein interaction database. To study the function of interactors, we used a triple co-transfection system to overexpress LRIG1 and PDGFRA and downregulate endogenous levels of interactors by short hairpin RNAs (shRNAs), simultaneously. This analysis demonstrated that CNPY3, CNPY4, GAL3ST1, GML, HLA-DRA, LRIG2, LRIG3, LRRC40, PON2, RAB4A, and ZBTB16 were important for the PDGFRA-downregulating function of LRIG1.

To investigate the clinical significance of LRIG1 copy number alterations (CNAs) in breast cancer, we used droplet digital PCR (ddPCR) to analyze 423 breast cancer tumors. We found that LRIG1 CNAs were significantly different in steroid-receptor-positive vs steroid-receptor-negative tumors and in ERBB2-amplified vs ERBB2-non-amplified tumors. In the whole cohort, patients with LRIG1 loss or gain had a worse metastasis-free survival than patients with normal LRIG1 copy numbers, however, among the early-stage breast cancer subgroup, this difference was not significant. 

In summary, Lrig1 behaved like a haploinsufficient tumor suppressor gene in malignant glioma, whereas Lrig2 appeared to promote malignant glioma. Our functional analysis of LRIG1 interactome uncovered several unanticipated and novel proteins that might be important for the regulation of receptor tyrosine kinases by LRIG1. LRIG1 CNAs predicted metastasis-free survival time in breast cancer. Hopefully, our findings might lead to a better understanding of the regulation of growth factor signaling and its importance in cancer biology and prognosis.

The leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family has three members, LRIG1, LRIG2, and LRIG3, that encode three structurally similar transmembrane proteins. LRIG1 is a receptor tyrosine kinase regulator, tumor suppressor, and stem cell marker in the skin, intestine, and brain. LRIG2 and LRIG3 have been less studied but shown to interact with LRIG1. The different roles and mechanisms of action of LRIG proteins have not yet been fully elucidated. In Caenorhabditis elegans (C. elegans), the LRIG homolog SMA-10 regulates bone morphogenetic protein (BMP) signaling; however, this function has not been demonstrated for mammalian LRIG proteins. In mice, the gene encoding the neurodevelopmental guidance cue netrin-1, Ntn1, interacts with Lrig3 in inner ear development. The physical interactions between LRIG proteins and other proteins are mostly unknown. 

Here, we describe an LRIG1-centered protein interaction network that regulates growth factor receptor levels. The LRIG1 interactome comprised LRIG2 and LRIG3 as well as many unanticipated proteins. 

An unbiased pathological examination of female mice with different Lrig3 genotypes (homozygous, heterozygous, or knockout) revealed a reduced incidence of spontaneous fatty liver and lymphocytic hyperplasia of the spleen in Lrig3-null mice. Female Lrig3-null mice also had a lower incidence of microvesicular cytoplasm in the liver after eight weeks on a high-fat diet. 

To further explore the molecular and physiological functions of LRIG proteins, we generated Lrig-null (Lrig1^-/-;Lrig2^-/-;Lrig3^-/-) mouse embryonic fibroblasts (MEFs), which displayed a deficiency in adipogenesis caused by impaired BMP signaling. LRIG1 and LRIG3, but not LRIG2, sensitized cells to BMP and rescued the adipogenesis deficiency in Lrig-null MEFs. In C. elegans, the LRIG homolog sma-10 was needed for proper lipid accumulation. By analyzing data from the UK Biobank and GENiAL cohort, we found that certain LRIG1 gene variants were associated with a higher body mass index (BMI) yet protected against type 2 diabetes. This effect was probably mediated by altered adipocyte morphology. 

CRISPR/Cas9-mediated ablation of Ntn1 revealed that the BMP-promoting function of LRIG1 and LRIG3 was opposed by netrin-1, which functioned as an inhibitor of BMP signaling via its receptor neogenin.

In summary, the present thesis describes a novel LRIG protein interaction network, the regulation of BMP signaling by LRIG proteins and netrin-1, and an important function of LRIG proteins in regulating fat metabolism with implications for human metabolic health.